1. Field of the Invention
The invention relates to polypeptides corresponding to soluble isoforms of human prolactin receptor and functional derivatives thereof. The invention also relates to recombinant DNA molecules encoding soluble human prolactin receptors, expression vectors carrying the recombinant DNA molecules and host cells carrying such expression vectors and capable of expressing a soluble human prolactin receptor.
2. Description of the Background Art
Two of the adenohypophysial peptide hormones, prolactin (PRL) and growth hormone (GH), are thought to have evolved from a common ancestral gene (Kelly et al., Recent Prog. Horm. Res. 48: 123-164, 1993). The receptors for PRL and GH (PRLR and GHR) show high homology to each other and belong to the GH/PRL/cytokine receptor superfamily. Thus, they are also thought to be cognates of a common ancestral gene (Kelly et al., 1993, supra). The signaling pathways of PRL and GH have been recently reported to involve a receptor-associated tyrosine kinase. The common structural features of the members of this superfamily are two pairs of cysteines and the tryptophan-serine (WS) motif in the extracellular domain. In the case of PRLR, there are five cysteines in the extracellular domain, and two pairs of cysteines and the WS motif have been shown to be important for ligand binding (Rozakis-Adcock et al., J. Biol. Chem. 266: 16472-16477, 1991; J. Biol. Chem. 267: 7428-7433, 1992).
The primary structure of the mature human GH receptor is a polypeptide of 620 amino acids (aa) with an extracellular hormone-binding domain of 246 aa, a single transmembrane region, and a cytoplasmic domain of 350 amino acid residues. The extracellular domain contains seven cysteine residues and five potential N-linked gylcosylation sites (Kelly et al., 1993, supra). While the structure of the mature PRL receptor from rat liver was deduced from CDNA to contain 291 amino acids with an extracellular region of 210 aa having five cysteine residues and three potential N-linked glycosylation sites, a single transmembrane region of 24 aa, and a relatively short cytoplasmic domain of 57 aa (Boutin et al, Cell 53: 69-77, 1988 represents a first class of receptor (short form), the structure of mature human PRLR containing 598 amino acid residues represents a second class of receptor (long form) (U.S. Pat. No. 4,992,378). Like mature rat liver GHR, this long form human PRLR has an extracellular region of 210 aa and a single transmembrane region, but a long cytoplasmic region.
Interestingly, the extracellular domain of human prolactin receptor was shown to be capable of binding both human prolactin and human growth hormone with nearly equal affinity (Boutin et al., Mol. Endocrinol. 3: 1455-1461, 1989). Cunningham et al., Science 250: 1709-1712 (1990) later reported that the binding of human growth hormone to the human prolactin receptor requires the presence of a zinc atom. In contrast, the human prolactin receptor does not require zinc in order to bind hPRL and it appears that while PRL- and GH-specific determinants on the human prolactin receptor overlap in certain areas, they are not identical (Rozakis-Adcock et al., 1992, supra).
Human GHR is known to possess several isoforms, including the GH-binding protein, secreted extracellular domain of the receptor, and GHRd3, which lacks exon 3 (Leung et al., Nature 330: 537-543, 1987; Urbanek et al., J. Biol. Chem. 268: 19025-19032, 1993). Furthermore, GH binding protein (GH-BP), which is a soluble short form of the liver GH receptor was identified in the serum of mouse (Peeters et al., Endocrinology 101: 1164-1179, 1977), rabbit (Ymer et al., Mol. Cell. Endocrinol. 41: 153-161, 1985), and human (Baumann et al., J. Clin. Endocrinol. Metab. 62: 134-141, 1986). There is amino acid identity of the amino-terminal sequences of the receptor and the binding protein (BP) (Leung et al., 1987).
Two independent mechanisms have been proposed for the production of the GH-BP: specific proteolysis of the membrane form of the receptor or translation from an alternatively spliced mRNA, produced from the same gene as the GH receptor. In man, cow, and rabbit, the serum GH-BP probably results from proteolytic cleavage of the receptor, because only a single mRNA transcript of 4.5 kb has been clearly identified by Northern analysis. In mouse and rat liver, two mRNAS of approximately 4.5 and 1-1.5 kilobases (kb) are expressed, which encode the membrane receptor and the GH-BP, respectively (Smith and Talamantes et al., J. Biol. Chem. 262: 2213-2219, 1987; Baumbach et al., Genes Dev. 3: 1199-1205, 1989). In rat and mouse, the GH-BP is distinguished by a short hydrophilic C-terminal extension that is not found in the membrane receptor. Using an antibody raised against a synthetic peptide containing the 17-aa hydrophilic sequence, it was clearly shown that the GH BP in rat serum is derived almost entirely from the GH BP mRNA and not from proteolytic processing of the GH receptor (Sadeghi et al., Mol. Cell. Endocrinol. 4: 1799-1805, 1990), probably as a result of alternative splicing of a primary transcript.
In the rat, a mutant form of the PRL receptor was identified in the Nb2 lymphoma cell line, which is dependent on PRL for growth and contains high-affinity PRL receptors (Gout et al., Cancer Res. 40: 2433-2436, 1980; Shiu et al., Endocrinology 113: 159-165, 1983). Biochemical studies have suggested that the receptor in these cells is different from the short and long forms already identified. Using the polymerase chain reaction of reverse-transcribed RNA and classical screening of a cDNA library, it was shown that the rat Nb2 PRL receptor is intermediate in size (393 aa) and appears to be due to a mutation in the PRL receptor gene, resulting in a loss of 594 bp in a region encoding a major portion of the cytoplasmic domain of the long form of the PRL receptor (Ali et al., 1991, supra). However, this mutant form of the rat PRL receptor still retains the transmembrane region and a portion of the cytoplasmic domain and is not a soluble isoform of PRLR. FIG. 1 shows a schematic presentation of the GH/PRL receptor family.
In a human breast cancer cell line, a cDNA obtained from a transcript was characterized and presumed to code for a soluble extracellular domain of the hPRL receptor where the transmembrane domain exon of hPRLR is deleted and results in a frameshift mutation after Ser.sup.204 and a stop codon after two additional residues, Ala.sup.205 and Trp.sup.206 (Fuh et al., J. Biol. Chem. 270: 13133-13137, 1995). This is the only hPRLR cDNA reported to be transcribed from an alternatively spliced form of hPRLR transcripts.
GH is known to promote water and electrolyte transport and calcium absorption in the gastrointestinal (GI) tract,(Lobie et al., 1990, supra and references cited therein). The proliferative functions of GH have been reported as well. Administration of GH restores the intestinal and gastric mucosal weight after hypophysectomy in the rat (Scow et al., Endocrinol. 77: 852-858, 1965) and is protective against gastric ulceration (Winawer et al., Arch. Intern. Med. 135: 569-572, 1975). Also, overexpression of bovine GH enhances growth of small intestine in the transgenic mice (Ulshen et al., Gastroenterology 104: 973-980, 1993), and the growth of fetal rat intestinal transplants is GH dependent (Cooke et al., Biol. Neonate 49: 211-218, 1986). More recently, using GH-deficient rats, Lobbie et al. (Lobie et al., Endocrinology 130: 3015-3024, 1992) clearly demonstrated that GH stimulates proliferation and enlargement of the gastric mucosa without significant alteration in cellular composition. Furthermore, they showed that GH administration results in stimulation of gastric mucosal intrinsic factor content in parietal and chief cells in the rat. It was reported that hypophysectomy results in a decreased level of plasma gastrin and in hypotrophy of the GI tract (Enochs et al., Gastroenterology 70: 727, 1976) and that GH exerts the opposite effects as well as an increase of the plasma gastrin level (Enochs et al., supra; Scow et al., supra). Thus, in addition to the effect of GH and its action via insulin-like growth factor I secretion, the trophic effect of GH on the GI tract is thought to be at least partially mediated by gastrin.
In contrast to GH, functions of PRL in the mammalian GI tract have not been well established. PRL acts on water and electrolyte transport as well as on calcium absorption (Dusanter-Fourt et al., 1992, supra, and references cited therein). PRL in the milk is transferred into the blood circulation of neonates and may modulate the development of neuroendocrine, reproductive, and immune systems (Grosvenor et al., Endocrine Rev. 14: 710-728, 1992). The intestinal mucosal mass is known to increase during lactation in the rat. This trophic effect of PRL was examined using PRL-treated adult rats, and it was reported that hyperprolactinemia did not result in such changes in villus height, crypt depth, or mucosal weight, although breast hyperplasia and increase in mammary pad weight did occur (Goodlad et al., Balliere's Clin. Gastroenterol. 4: 97-118, 1990). However, it was also reported that inhibition of PRL secretion by bromocriptine inhibits the increase in the intestinal weight (Goodlad et al., 1990, supra); thus, hormones such as steroids or GI tract trophic hormones should be considered along with PRL for such effects.
It is now widely accepted that PRL and GH immunomodulation such as the proliferation of lymphocytes from spleens or lymph nodes of ovariectomized rats, and increase in cytotoxic activities of natural killer cells (Kelly et al., 1993, supra). Although no severe clinical disturbance in immune function has been reported for GH- or PRL-deficient patients, PRL and GH are capable of restoring immune function in hypophysectomized animal models or genetic dwarf mice (Kelly et al., 1993, supra). In vitro studies have also shown direct effects of these hormones as immunostimulatory factors on lymphocytes (Kelly et al., 1993, supra). Furthermore, expression of PRL, GH, and their receptors in lymphocytes has been well documented (Kelly et al., 1993, supra; Pellegrini et al., Mol. Endocrinol. 6: 1023-1031, 1992). Considering the importance of the GI tract as an immunologic barrier against foreign pathogens, there two hormones may act on immune cells in this tissue. The fact that lymphocytes, and perhaps other GH or PRL target cells are capable of producing these hormones suggests that they should be also considered "growth factors", acting via classical paracrine or autocrine pathways.